Hepatic lipogenesis represents a crucial part of intermediary metabolism and is acutely regulated by endocrine factors and nutrients. The liver-specific and highly abundant microRNA-122 has emerged as a powerful regulator of lipogenesis in higher vertebrates, but little is known about its endocrine and nutritional regulation. In this study, we investigated the hypothesis that insulin regulates hepatic expression of omy-miRNA-122 isomiRNAs (omy-miRNA-122a and omy-miRNA-122b) by using in vivo and in vitro approaches. Since the hepatic insulin pathway and lipogenesis are acutely regulated by dietary macronutrient ratios in rainbow trout, we further investigated the effect of single meals with altered carbohydrate/protein ratio and lipid/protein ratio on the postprandial expression of omy-miRNA-122 isomiRNAs. Insulin acutely induced omy-miRNA-122b expression in vivo and in vitro. Conversely, a single meal with increased lipid to protein ratio acutely decreased expression of both omy-miRNA-122 isomiRNAs. As a direct proof of lipogenic effects of miRNA-122 is currently still lacking in fish, we investigated the correlated expression between omy-miRNA-122 isomiRNAs and the rate-limiting lipogenic gene fas, an indirect target gene of miRNA-122 in mammals. Our results show a significant positive correlation of omy-miRNA-122b and fas, consistent with a potential evolutionary conserved role for miRNA-122 in the regulation of postprandial lipogenesis in trout